Stimulation of an immune response is dependent upon the presence of antigens recognized as foreign by the host immune system. The discovery of the existence of tumor associated antigens has now raised the possibility of using a host's immune system to intervene in tumor growth. Various mechanisms of harnessing both the humoral and cellular arms of the immune system are currently being explored for cancer immunotherapy.
Specific elements of the cellular immune response are capable of specifically recognizing and destroying tumor cells. The isolation of cytotoxic T cells (CTL) from tumor-infiltrating cell populations or from peripheral blood suggests that such cells play an important role in natural immune defenses against cancer (Cheever et al., Annals N.Y. Acad. Sci. 1993 690:101-112; Rosenberg S A. Shedding light on immunotherapy for cancer. N Engl J. Med. 2004 Apr. 1; 350(14):1461-3.). CD8+ T-cells (TCD8+) in particular, which recognize Class I molecules of the major histocompatibility complex (MHC)-bearing peptides of usually 8 to 10 residues derived from proteins located in the nucleus or the cytosol, or from defective ribosomal proteins (DRIPs), play an important role in this response. DRIPs are an essential source-for peptides and constitute products of incomplete translation at the ribosomes and have-first been described by the group of J. Yewdell (Schubert U, Anton L C, Gibbs J, Norbury C C, Yewdell J W, Bennink J R. Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature. 2000 Apr. 13; 404(6779):770-4). The MHC molecules of the human are also designated as human leukocyte-antigens (HLA).
There are two major classes of MHC-molecules that can be recognized by T-cells bearing T cell receptors. MHC-1-molecules that can be found on most cells having a nucleus that present peptides that result from proteolytic cleavage of endogenous proteins and larger peptides. MHC-II-molecules can be found on professional antigen presenting cells (APC), such as Macrophages, Dendritic Cells, on B cells, on endothelial cells and on altered cells of tumors and tumor stroma which, under normal circumstances, do not express MHC class II-molecules on their cell surfaces, and present either peptides stemming exogenous proteins that are taken up by APCs during the course of endocytosis, or that otherwise enter the MHC class II compartment (MIIC) and are subsequently processed and loaded onto MHC class II complexes. Complexes of peptide and MHC-I are recognized by CD8+-positive cytotoxic T-lymphocytes, whereas complexes of peptide and MHC-II are recognized by CD4+-helper-T-cells (generally described in Immunobiology by Charles A., Jr. Janeway, Paul Travers, Mark Walport, Mark J. Shlomchik).
For a peptide to trigger (elicit) a cellular immune response, it must bind to an MHC-molecule. This process is dependent on the allele of the MHC-molecule and specific polymorphisms of the amino acid sequence of the peptide. MHC-class-1-binding peptides are usually 8-10 residues in length and contain two conserved residues (“anchor”) in their sequence which interact with the corresponding binding groove of the MHC-molecule (see 2nd listing as published in Immunogenetics (Rammensee H, Bachmann J, Emmerich N P, Bachor O A, Stevanovic S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999 November; 50(3-4): 213-9).
There are now numerous examples of both mouse and human TCD8+ that specifically recognize tumor cells and have therapeutic activity after adoptive transfer, in some cases inducing complete remission. However, despite the potential for T cells to eradicate tumors, it is obvious from the progressive growth of most cancers that many tumors escape recognition by TCD8+ in vivo. Though a variety of tumors have been found to be immunogenic, stimulation of an effective anti-tumor immune response has been difficult to demonstrate. Latest evidence shows that immunizations can lead to strong T cell responses against tumorassociated peptides (Speiser D E, Lienard D, Rufer N, Rubio-Godoy V, Rimoldi D, Lejeune F,
Krieg A M, Cerottini J C, Romero P. Rapid and strong human CD8+ T-cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleot 7909. J Clin Invest. 2005 March; 115(3):739-46. Schag K, Schmidt S M, Muller M R, Weinschenk T, Appel S, Weck M M, Grunebach F, Stevanovic S, Rammensee H G, Brossart P. Identification of C-met oncogene as a broadly expressed tumor-associated antigen recognized by cytotoxic T-lymphocytes. Clin Cancer Res. 2004 Jun. 1; 10(11):3658-66.
Antigens that are recognized by the tumor specific cytotoxic T-lymphocytes, that is, their epitopes, can be molecules derived from all protein classes, such as enzymes, receptors, transcription factors, etc. A comprehensive listing of peptides binding to or eluted from MHC class I or class II molecules can be found on www.syfpeithi.org. Furthermore, tumor associated antigens, for example, can also be present in tumor cells only, for example as products of mutated genes, e.g. MHC class I ligands, which function as T cell epitopes, from K-ras, BCR-abl and mutated p53. Another important class of tumor associated antigens are tissue-specific structures, such as CT (“cancer testis”)-antigens that are expressed in different kinds of tumors and in healthy tissue of the testis. Other tumor associated peptides binding to MHC molecules stem from genes, which are expressed in higher copy numbers in cancer cells compared to healthy cells of the same organ or tissue, as well as compared to healthy cells from other tissues, e.g. c-met (See Schag K, Schmidt S M, Muller M R, Weinschenk T, Appel S, Weck M M, Grunebach F, Stevanovic S, Rammensee H G, Brossart P. Identification of Cmet oncogene as a broadly expressed tumor-associated antigen recognized by cytotoxic Tlymphocytes. Clin Cancer Res. 2004 Jun. 1; 10(11):3658-66). Other tumor-associated peptides stem from antigens that are retained in tumor cells and not secreted (e.g., proteins from the mucin gene family). Other sources can be aberrant transcripts (frameshift) or peptides from junction sites of post-translational protein-protein fusions. A comprehensive listing of tumor associated antigens described in the scientific literature can be found on www.cancerimmunity.org.
Various tumor associated antigens have been identified. Further, much research effort is being expended to identify additional tumor associated antigens. Some groups of tumor associated antigens, also referred to in the art as tumor specific antigens, are tissue specific. Examples include, but are not limited to, tyrosinase for melanoma, PSA and PSMA for prostate cancer and chromosomal cross-overs such as bcr/abl in lymphoma. However, many tumor associated antigens that have been identified occur in multiple tumor types, and some, such as oncogenic proteins and/or tumor suppressor genes (tumor suppressor genes are, for example reviewed for renal cancer in Linehan W M, Walther M M, Zbar B. The genetic basis of-cancer of the kidney. J Urol. 2003 December; 170(6 Pt 1):2163-72), which-actually cause the transformation event, occur in nearly all tumor types. A more general review of genetic causes of human cancer can be found in The Genetic Basis of Human Cancer by Bert Vogelstein, Kenneth W. Kinzler, 2002). For example, normal cellular proteins that control cell growth and differentiation, such as p53 (which is an example for a tumor suppressor gene), ras, c-met, myc, pRB, VHL, and HER-2/neu, can accumulate mutations resulting in upregulation of expression of these gene products thereby making them oncogenic (McCartey et al. Cancer Research 1998 15:58 2601-5; Disis et al. Ciba Found. Symp. 1994 187:198-211). These mutant proteins can be the target of a tumor specific immune response in multiple types of cancer.
The oncofoetal antigen-immature laminin receptor protein (OFA/iLRP) is widely expressed in many types of human tumors including hematopoietic malignancies (Rohrer J W, Barsoum A L, Coggin J H Jr. The development of a new universal tumor rejection antigen expressed on human and rodent cancers for vaccination, prevention of cancer, and anti-tumor therapy. Mod Asp Immunobiol. 2001; 5: 191-195. Barsoum A L, Rohrer J W, Coggin J H. 37 kDa oncofoetal antigen is an autoimmunogenic homologue of the 37 kDa laminin receptor precursor. Cell Mol Biol Lett. 2000; 19: 5535-5542. Castronovo V. Laminin receptors and laminin-binding proteins during tumor invasion and metastasis. Invasion Metas. 1993; 13: 1-30. Coggin J H Jr, Barsoum, A L, Rohrer J W. Tumors express both unique TSTA and crossprotective 44 kDa oncofetal antigen. Immunol Today. 1998; 19, 405-408. Coggin J H Jr, Barsoum A L, Rohrer J W. 37 kilo Dalton oncofetal antigen protein and immature laminin receptor protein are identical, universal T-cell inducing immunogens on primary rodent and human cancers. Anticancer Res. 1999; 19, 5535-5542)1 but is not present in normal adult differentiated tissues. OFA-iLRP can be specifically recognized by both T and B lymphocytes, making it an attractive target molecule for vaccination approaches in several cancer entities. Utilizing dendritic cells (DC) transfected with OFA-iLR-coding RNA, tumor-specific T cell responses against hematopoietic target cells could be generated both in vitro and in vivo (Siegel S, Wagner A, Kabelitz D et al. Coggin, J. Jr., Barsoum, A., Rohrer, J., Schmitz, N., Zeis, M. Induction of cytotoxic T cell responses against the oncofoetal antigenimmature laminin receptor for the treatment of hematological malignancies. Blood 2003; 102, 4416-4423.
U.S. Pat. No. 6,753,314 describes a purified protein complex comprising a first polypeptide and a second polypeptide, wherein said complex comprises the amino acid sequences of a first polypeptide (SMI1, SEQ ID NO: 359), and a second polypeptide (BAST, SEQ ID NO: 518), denoted as ProPair 267a-267b.
U.S. Pat. No. 4,861,710 discloses a clone comprising a recombinant cDNA clone for encoding cell surface receptor for laminin as well as respective probes.
For the proteins to be recognized by the cytotoxic T-lymphocytes as tumor-specific antigen, and to be useful in a therapy, particular prerequisites must be fulfilled. The antigen should be expressed mainly by tumor cells and not by normal healthy tissues or in rather small amounts. It is furthermore desirable; that the respective antigen is not only present in one type of tumor, but also present high concentrations (e.g. copy numbers per cell). The presence of epitopes in the amino acid sequence of the antigen is essential, since such peptide (“immunogenic peptide”) that is derived from a tumor associated antigen should lead to an in vitro or in vivo T-cell-response.
Until now, numerous strategies to target antigens into the class II processing pathway have been described. It is possible to incubate antigen presenting cells (APCs) with the antigen of interest in order to be taken up and processed (Chaux, P., Vantomme, V., Stroobant, V., Thielemans, K., Corthals, J., Luiten, R., Eggermont, A. M., Boon, T. & van der, B. P. (1999) J. Exp. Med. 189, 767-778). Other strategies use fusion proteins that contain lysosomal target sequences. Expressed in APCs, such fusion proteins direct the antigens into the class II processing compartment (Marks, M. S., Roche, P. A., van Donselaar, E., Woodruff, L., Peters, P. J. & Bonifacino, J. S. (1995) J. Cell Biol. 131, 351-369, Rodriguez, F., Harkins, S., Redwine, J. M., de Pereda, J. M. & Whitton, J. L. (2001) J. Virol. 75, 10421-10430). Also, special liposomal formulations for the delivery of peptides and other active pharmaceutical ingredients to pAPC have been developed ((Walter S, Herrgen L, Schoor O, Jung G, Wernet D, Buhring H J, Rammensee H G, Stevanovic S. Cutting edge: predetermined avidity of human CD8 T cells expanded on calibrated MHC/anti-CD28-coated microspheres. J Immunol. 2003 Nov. 15; 171(10):4974-84. Another method involves external loading of MHC molecules of pAPC in vitro or in vivo. In this setting, APCs are incubated with an excess of peptides in cell culture media, leading to competition for binding to MHC molecules on the surface of the APC.
T-helper cells play an important role in orchestrating the effector function of CTLs in anti-tumor immunity. T-helper cell epitopes that trigger a T-helper cell response of the Th1 type support effector functions of CD8+ Killer T-cells, which include cytotoxic functions directed against tumor cells displaying tumor-associated peptide/MHC complexes on their cell surfaces. In this way tumor-associated T-helper cell peptide epitopes, alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients of vaccine compositions which stimulate anti-tumor immune responses.
The major task in the development of a tumor vaccine is therefore the identification and characterization of novel tumor associated antigens and immunogenic T-helper epitopes derived therefrom, which can be recognized by CD4+ CTLs. It is therefore an object of the present invention, to provide novel amino acid sequences for such peptides that have the ability to bind to a molecule of the human major histocompatibility complex (MHC) class-I and trigger T cell responses against cells bearing the peptides in conjunction with MHC molecules on their cell surfaces.